The genetic treatment of X-linked severe combined immunodeficiency (XSCID) and most immune and hematological disorders will require the transduction of pluripotent, self-renewing hematopoietic stem cells (HSCs) rather than their progenitors in order to achieve enduring production of genetically corrected cells and durable immune reconstitution. Gene-corrected HSCs will have to compete not only with the defective host cells but also with a significant number of uncorrected HSCs depending upon the transduction efficiency. In XSCID, the selective advantage of T cells expressing a normal common gamma chain (gamma c) gene means that transduction of small numbers of immature progenitor cells may produce clinically significant numbers of mature T cells. However, the lack of a selective advantage in the B cell lineage means that reconstitution of normal B cells may depend on transduction of a large number of immature progenitors and/or HSCs. The results of the recent successful retroviral gene therapy trial for human XSCID suggest that the number of transduced cells will be important for the quality and durability of immune reconstitution following treatment. Although all successfully treated patients developed normal levels of T cells following treatment, they developed few, if any, gene-corrected B cells. However the oldest patient is showing decline in the number of T cells raising questions of the durability of T cell reconstitution. In addition, one of the patients recently developed a T cell leukemia as a result of the gene therapy. The overall hypothesis of this competitive renewal is that increasing the number of genetically corrected HSCs and/or progenitors will result in improved quality and durability of immune reconstitution. The general prediction is that the vectors that give the highest rate of HSC transduction will produce more B cells and better humoral function as well as produce a more durable T cell reconstitution. Therefore, the major focus of the proposed studies is to evaluate strategies for improving gene delivery to increase the number of gene-corrected cells. Special emphasis will be placed upon lentiviral vectors that, unlike retroviral vectors, can transduce non-cycling cells that should result in increased numbers of gene-corrected HSCs. We will also evaluate strategies for enhancing the selective advantage of gene corrected cells using either nonmyeloablative conditioning or in vivo selection of genetically corrected cells. Since higher transduction efficiencies and/or enhanced engraftment of transduced cells may lead to an overall higher number of transgene insertions increasing the potential risk of insertional mutagenesis, we will perform longitudinal integration site analyses to determine whether clonality changes over time and for risk assessment of side effects related to transgene integration and expression. Canine XSCID has an identical immunologic phenotype as human XSCID making it an ideal large-animal model in which to perform these studies.